Many processes require a specific temperature in such a way that the efficacy of the process or a particular step in the process is dependent on the execution of the process/step at a specific temperature. Monitoring of the temperature of the environment of the process/step is necessary for that the environment can either be heated or cooled to maintain the temperature at the desired temperature as necessary. Continued monitoring of the environment temperature is required so that the temperature can be maintained at the desired level by heating or cooling, as necessary. The same considerations apply when different temperatures are required for further steps in a process.
One example for a process which requires specific temperatures is the polymerase chain reaction (PCR) which involves multiple cycles. The PCR is a scientific technique in molecular biology, which amplifies polynucleotide sequences each time a cycle is completed. The technique of PCR is well known and is described in many books, including, PCR: A Practical Approach M. J. McPherson, et al., IRL Press (1991), PCR Protocols: A Guide to Methods and Applications by Innis, et al., Academic Press (1990), and PCR Technology: Principals and Applications for DNA Amplification H. A. Erlich, Stockton Press (1989). PCR is also described in many U.S. patents, including U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; 4,889,818; 5,075,216; 5,079,352; 5,104,792; 5,023,171; 5,091,310; and 5,066,584.
Each PCR cycle usually comprises three basic discrete temperature steps, a denaturation step, an annealing step and an extension step. Denaturation of DNA typically takes place at around 90 to 95° C., annealing a primer to the denatured DNA is typically performed at around 40 to 60° C., and the step of extending the annealed primers with a polymerase is typically performed at around 70 to 75° C. Therefore, during a PCR cycle the temperature of the reaction mixture must be varied, and varied many times during a multicycle PCR experiment. The efficacy of each step is strongly dependent on the temperature at which each step is carried out. The PCR technique has a wide variety of biological applications, including for example, DNA sequence analysis, probe generation, cloning of nucleic acid sequences, site-directed mutagenesis, detection of genetic mutations, diagnoses of viral infections, molecular “fingerprinting” and the monitoring of contaminating microorganisms in biological fluids and other sources. The PCR is usually carried out in a laboratory apparatus called thermal cycler or PCR cycler.
In addition to PCR, other in vitro amplification procedures, including ligase chain reaction as disclosed in U.S. Pat. No. 4,988,617 to Landegren and Hood, are known and advantageously used in the prior art. More generally, several important methods known in the biotechnology arts, such as nucleic acid hybridization and sequencing, are dependent upon changing the temperature of solutions containing sample molecules in a controlled fashion. Conventional techniques rely on use of individual wells or tubes cycled through different temperature zones. For example, a number of thermal “cyclers” used for DNA amplification and sequencing are disclosed in the prior art in which a temperature controlled element or “block” holds a reaction mixture, and wherein the temperature of the block is varied over time. One advantage of these devices is that a relatively large number of samples can be processed simultaneously, e.g. 96 well plates are commonly employed.
Different designs of such thermal cyclers are known. E.g. devices are available for the thermal cycling of multiple samples, typically for the amplification of DNA. A common format of such devices is the inclusion of a block of heat conductive material which has a plurality of channels or cavities therein for receiving vessels—such as reaction tubes or plates—in which the desired reactions are executed. Monitoring of temperature is relatively easy in such devices since a temperature probe can be associated with the block.
However, such block devices suffer various drawbacks, e.g. in that they are relatively slow in cycling the reaction mixtures, they are relatively energy intensive to operate and detection of the reaction mixture in situ is difficult. In an effort to avoid several of these disadvantages, other thermal cyclers have been developed in which a plurality of containers for holding reaction mixture(s) are supported on a rotatable carousel rotatably mounted within a chamber adapted to be heated and cooled.
Devices for thermal cycling of reaction mixtures are also known in which the reaction vessels are held in a rotor which is rotated in a controlled temperature environment such as an insulated chamber containing the rotor (also known as “Rotor-Gene”). Temperature cycling is effected by heating and cooling of the environment. The carousel or rotor has apertures, receptacles or slots for the containers, vials or tubes. Rotors are known, which have 60 or 72 receptacles for a corresponding number of vials or tubes. Such a device is disclosed, for example, in International Patent Application No. PCT/AU98/00277 (Publication No. WO 98/49340). Since reaction mixtures are rotated in the PCT/AU99/00277 device, it is difficult to accurately measure the temperature of a reaction mixture. Regardless of how well the temperature of the rotor environment is controlled, there can be a temperature difference between a reaction mixture and the environment per se. Since accurate temperature control is essential for most thermal cycling reactions, knowledge of the actual temperature of the reaction mixtures, i.e. the liquid in the reaction tube is important. Compensation can then be made in the environmental temperature management program to give a desired temperature in the actual reaction tube. However, the dynamic thermal behaviour of a volume of an aqueous solution within a rotating system is not easy to monitor. Sensors with cables will introduce an additional thermal capacity. Further, the cables will form an additional thermal bridge, which may also influence the measurement of the temperature. Thus, while it is generally easy to monitor the temperature of the overall environment of a process, it is not always easy to monitor the temperature of process micro-environments.
An important aspect of the operation of respective thermal cyclers thus is the accurate control of the temperature of the contents of reaction vessels. This is equivalent to how accurately the temperature of the air surrounding the reaction vessels is controlled. However, the temperature within a vessel may not necessarily be the same as the chamber temperature as sensed by the Rotor-Gene's temperature control equipment, so compensation has to be made in that equipment.
Known from WO 03/102522 A1 is a system and a method for the optical calibration of the temperature of a reaction in a vessel. A relationship between the detectable chamber temperature and the temperature in the reaction vessel is obtained in the calibration procedure. During calibration a transparent vial having a luminophore-containing component layered over or under a thermochromic liquid crystal (TLC) component is used. The system comprising the luminophore-containing component and the TLC component is irradiated by a light source and any luminescence emitted by the luminophore-containing component is detected by a detector while recording the temperature of said environment. The obtained data is used to provide a temperature calibration curve with regard to chamber temperature versus reaction vial temperature. Thereby, it can be controlled/verified, that the temperature control of a respective device works accurately. For this purpose, the combination of luminophore and TLC is provided in a micro-environment (reaction vial) and e.g. a PCR profile is run in the course of which different discrete temperatures must be established in the micro-environment. The transition in the luminescence of the TLC/luminophore combination is recorded. Based on the assumed transition temperature of said combination, the actual temperature that occurred in the micro-environment is deducted/determined and compared to the temperature that was supposed to be achieved if the temperature control worked properly. If deviations are found, appropriate adjustment in the temperature control of the device are performed until the actual temperature in the micro-environment that is determined by the TLC/luminophore combination matches sufficiently well the expected temperature.
The accuracy of the above mentioned methods and systems leave room for improvement. Further, when using optical measurement for determining the temperature an optical drift might occur which influences the measurement. For example in case that the luminous flux of the illuminating source varies with time, the detected intensity of the optical detector varies. Further, the irradiating source, namely the luminous flux, might be influenced by the temperature of the irradiating source, and this optical drift will directly result in a biased temperature measurement. This poses a problem in particular with thermal cyclers which have no reference channel for compensating drifts in the optical system.
The present invention seeks to overcome or ameliorate at least one of the disadvantages of the above mentioned prior art or to provide a useful alternative.